Lazy neutrophils - a lack of DGAT1 reduces the chemotactic activity of mouse neutrophils.

BACKGROUND: Neutrophils are key players in the innate immune system, actively migrating to sites of inflammation in the highly energetic process of chemotaxis. In this study, we focus on the role of acyl-CoA: diacylglycerol acyltransferase 1 (DGAT1), an enzyme that catalyzes the synthesis of triglycerides, the major form of stored energy, in neutrophil chemotaxis. METHODS AND RESULTS: Using a mouse model of psoriasis, we show that DGAT1-deficiency reduces energy-demanding neutrophil infiltration to the site of inflammation, but this inhibition is not caused by decreased glycolysis and reduced ATP production by neutrophils lacking DGAT1. Flow cytometry and immunohistochemistry analysis demonstrate that DGAT1 also does not influence lipid accumulation in lipid droplets during inflammation. Interestingly, as has been shown previously, a lack of DGAT1 leads to an increase in the concentration of retinoic acid, and here, using real-time PCR and publicly-available next-generation RNA sequencing datasets, we show the upregulation of retinoic acid-responsive genes in Dgat1KO neutrophils. Furthermore, supplementation of WT neutrophils with exogenous retinoic acid mimics DGAT1-deficiency in the inhibition of neutrophil chemotaxis in in vitro transwell assay. CONCLUSIONS: These results suggest that impaired skin infiltration by neutrophils in Dgat1KO mice is a result of the inhibitory action of an increased concentration of retinoic acid, rather than impaired lipid metabolism in DGAT1-deficient mice.

Alternative splicing is not a key source of chemerin isoforms diversity.

BACKGROUND: Chemerin is a chemoattractant protein with adipokine and antimicrobial properties encoded by the retinoic acid receptor responder 2 (RARRES2) gene. Chemerin bioactivity largely depends on carboxyl-terminal proteolytic processing that generates chemerin isoforms with different chemotactic, regulatory, and antimicrobial potentials. While these mechanisms are relatively well known, the role of alternative splicing in generating isoform diversity remains obscure. METHODS AND RESULTS: Using rapid amplification of cDNA ends (RACE) PCR, we determined RARRES2 transcript variants present in mouse and human tissues and identified novel transcript variant 4 of mouse Rarres2 encoding mChem153K. Moreover, analyses of real-time quantitative PCR (RT-qPCR) and publicly-available next-generation RNA sequencing (RNA-seq) datasets showed that different alternatively spliced variants of mouse Rarres2 are present in mouse tissues and their expression patterns were unaffected by inflammatory and infectious stimuli except brown adipose tissue. However, only one transcript variant of human RARRES2 was present in liver and adipose tissue. CONCLUSION: Our findings indicate a limited role for alternative splicing in generating chemerin isoform diversity under all tested conditions.

Antimicrobial Properties of a Peptide Derived from the Male Fertility Factor kl2 Protein of Drosophila melanogaster.

Antimicrobial peptides (AMPs) are important components of innate immunity. Here, we report the antimicrobial properties of a peptide derived from the Male fertility factor kl2 (MFF-kl2) protein of Drosophila melanogaster, which was identified as a functional analog of the mammalian antibacterial chemerin-p4 peptide. The antimicrobial activity of multifunctional chemerin is mainly associated with a domain localized in the middle of the chemerin sequence, Val66-Pro85 peptide (chemerin-p4). Using bioinformatic tools, we found homologs of the chemerin-p4 peptide in the proteome of D. melanogaster. One of them is MFF-p1, which is a part of the MFF kl2 protein, encoded by the gene male fertility factor kl2 (kl-2) located on the long arm of the Y chromosome. The second detected peptide (Z-p1) is a part of the Zizimin protein belonging to DOCK family, which is involved in cellular signaling processes. After testing the antimicrobial properties of both peptides, we found that only MFF-p1 possesses these properties. Here, we demonstrate its antimicrobial potential both in vitro and in vivo after infecting D. melanogaster with bacteria. MFF-p1 strongly inhibits the viable counts of E. coli and B. subtilis after 2 h of treatment and disrupts bacterial cells. The expression of kl-2 is regulated by exposure to bacteria and by the circadian clock.

Expression of antimicrobial peptide genes oscillates along day/night rhythm protecting mice skin from bacteria.

Antimicrobial peptides (AMPs) are important components of the innate immune system and are involved in skin protection against environmental insults and in wound healing. Herein, we assessed the gene expression of chemerin (Rarres2), cathelicidin CRAMP (Camp), and three ß-defensins (Defb1, Defb3, and Defb14) in mouse skin during light/dark cycle (LD 12:12) and constant darkness (DD). Next, we examined the survival of bacteria applied on the skin at specific times during the day. We found that the expression of Rarres2, Camp, and Defb1 was the highest at 4 h after the beginning of darkness, during high activity of mice. These rhythms, however, were not maintained under DD in the skin but were present in the liver. This indicated that in the case of skin, a circadian input was masked by daily changes of light in the environment. In contrast, Defb3 and Defb14 showed the highest mRNA levels when the mice slept, and these rhythmic mRNA oscillations were maintained under DD. This shows that Rarres2, Camp, and Defb1 levels in the skin are correlated with high locomotor activity in mice and they are controlled by daily changes of light and dark. Alternatively, oscillations in the mRNA levels of Defb3 and Defb14 seem to protect skin and heal wounds during sleep. These rhythms are maintained under DD, indicating that they are regulated by a circadian clock. Our study suggests that daily AMP expression affects the survival of bacteria on the surface of skin, which depends on the phase of AMP cycling.

Molecular Mechanisms of ZC3H12C/Reg-3 Biological Activity and Its Involvement in Psoriasis Pathology.

The members of the ZC3H12/MCPIP/Regnase family of RNases have emerged as important regulators of inflammation. In contrast to Regnase-1, -2 and -4, a thorough characterization of Regnase-3 (Reg-3) has not yet been explored. Here we demonstrate that Reg-3 differs from other family members in terms of NYN/PIN domain features, cellular localization pattern and substrate specificity. Together with Reg-1, the most comprehensively characterized family member, Reg-3 shared IL-6, IER-3 and Reg-1 mRNAs, but not IL-1ß mRNA, as substrates. In addition, Reg-3 was found to be the only family member which regulates transcript levels of TNF, a cytokine implicated in chronic inflammatory diseases including psoriasis. Previous meta-analysis of genome-wide association studies revealed Reg-3 to be among new psoriasis susceptibility loci. Here we demonstrate that Reg-3 transcript levels are increased in psoriasis patient skin tissue and in an experimental model of psoriasis, supporting the immunomodulatory role of Reg-3 in psoriasis, possibly through degradation of mRNA for TNF and other factors such as Reg-1. On the other hand, Reg-1 was found to destabilize Reg-3 transcripts, suggesting reciprocal regulation between Reg-3 and Reg-1 in the skin. We found that either Reg-1 or Reg-3 were expressed in human keratinocytes in vitro. However, in contrast to robustly upregulated Reg-1 mRNA levels, Reg-3 expression was not affected in the epidermis of psoriasis patients. Taken together, these data suggest that epidermal levels of Reg-3 are negatively regulated by Reg-1 in psoriasis, and that Reg-1 and Reg-3 are both involved in psoriasis pathophysiology through controlling, at least in part different transcripts.

The antimicrobial activity of chemerin-derived peptide p4 requires oxidative conditions.

Chemerin is a leukocyte attractant, adipokine, and antimicrobial protein abundantly produced in the skin epidermis. Despite the fact that most of the bactericidal activity present in human skin exudates is chemerin-dependent, just how chemerin shapes skin defenses remains obscure. Here we demonstrate that p4, a potent antimicrobial human chemerin peptide derivative, displays killing activity against pathogenic methicillin-resistant Staphylococcus aureus strains and suppresses microbial growth in a topical skin infection model. Mechanistically, we show that p4 homodimerization is required for maximal bactericidal activity and that an oxidative environment, such as at the skin surface, facilitates p4 disulfide bridge formation, required for the dimerization. p4 led to rapid damage of the bacterial internal membrane and inhibited the interaction between the membranous cytochrome bc1 complex and its redox partner, cytochrome c These results suggest that a chemerin p4-based defense strategy combats bacterial challenges at the skin surface.

Metagenomic Studies in Inflammatory Skin Diseases.

Next-generation sequencing (NGS) technologies together with an improved access to compute performance led to a cost-effective genome sequencing over the past several years. This allowed researchers to fully unleash the potential of genomic and metagenomic analyses to better elucidate two-way interactions between host cells and microbiome, both in steady-state and in pathological conditions. Experimental research involving metagenomics shows that skin resident microbes can influence the cutaneous pathophysiology. Here, we review metagenome approaches to study microbiota at this barrier site. We also describe the consequences of changes in the skin microbiota burden and composition, mostly revealed by these technologies, in the development of common inflammatory skin diseases.

The role of chemerin and its antibacterial derivatives in maintaining epithelial barrier function / Rola chemeryny i jej antybakteryjnych pochodnych w utrzymaniu funkcji barierowych nablonka.

The epithelial tissues have continuous contact with external environment, including pathogenic microorganisms. Endogenous antimicrobial proteins and peptides produced by epithelial cells play a key role in controlling microbial burden and composition, either directly, or by engaging immune cells. These include active derivatives of multifunctional protein chemerin, which is equipped with both antimicrobial and chemotactic function. Given an increasing number of infections caused by antibiotic-insensitive microorganisms, such as methicillin- resistant S. aureus (MRSA), it is important to fully understand how these epithelia-associated microorganisms are controlled at barrier sites, including skin and oral cavity. Chemerin-derived synthetic peptide 4 (p4) covering central Val66-Pro85 chemerin sequence exhibits broad range of antimicrobial activity against skin- and oral cavity- associated bacteria, including MRSA strains, suggesting its therapeutic potential for bacteria-mediated barrier organs pathologies. In this article we present the overview of protective functions of chemerin and chemerin-derived peptides in the epithelial tissues.

Role of myeloid regulatory cells (MRCs) in maintaining tissue homeostasis and promoting tolerance in autoimmunity, inflammatory disease and transplantation.

Myeloid cells play a pivotal role in regulating innate and adaptive immune responses. In inflammation, autoimmunity, and after transplantation, myeloid cells have contrasting roles: on the one hand they initiate the immune response, promoting activation and expansion of effector T-cells, and on the other, they counter-regulate inflammation, maintain tissue homeostasis, and promote tolerance. The latter activities are mediated by several myeloid cells including polymorphonuclear neutrophils, macrophages, myeloid-derived suppressor cells, and dendritic cells. Since these cells have been associated with immune suppression and tolerance, they will be further referred to as myeloid regulatory cells (MRCs). In recent years, MRCs have emerged as a therapeutic target or have been regarded as a potential cellular therapeutic product for tolerance induction. However, several open questions must be addressed to enable the therapeutic application of MRCs including: how do they function at the site of inflammation, how to best target these cells to modulate their activities, and how to isolate or to generate pure populations for adoptive cell therapies. In this review, we will give an overview of the current knowledge on MRCs in inflammation, autoimmunity, and transplantation. We will discuss current strategies to target MRCs and to exploit their tolerogenic potential as a cell-based therapy.

The role of metalloproteinase ADAM17 in regulating ICOS ligand-mediated humoral immune responses.

Immune cells regulate cell surface receptor expression during their maturation, activation, and motility. Although many of these receptors are regulated largely at the level of expression, protease-mediated ectodomain shedding represents an alternative means of refashioning the surface of immune cells. Shedding is largely attributed to a family of a disintegrin and metalloprotease domain (ADAM) metalloproteases, including ADAM17. Although ADAM17 is well known to contribute to the innate immune response, mainly by releasing TNF-α, much less is known about whether/how this metalloprotease regulates adaptive immunity. To determine whether ADAM17 contributes to regulating adaptive immune responses, we took advantage of ADAM17 hypomorphic (ADAM17(ex/ex)) mice, in which ADAM17 expression is reduced by 90-95% compared with wild-type littermates. In this study, we show that that ADAM17 deficiency results in spleen and lymph node enlargement, as well as increased levels of Ag-specific class-switched Ig production following immunization with OVA together with anti-CD40 mAbs and polyinosinic-polycytidylic acid. Moreover, we demonstrate that the costimulatory ligand ICOS ligand (ICOSL) is selectively downregulated on the surface of B cells in an ADAM17-specific manner, although it is not proteolitically processed by recombinant ADAM17 in vitro. Finally, we show that higher cell surface levels of ICOSL in ADAM17(ex/ex) mice may contribute to the development of excessive Ab responses. Therefore, our data suggest a functional link between ADAM17 and ICOSL in controlling adaptive immune responses.

The clinically used photosensitizer Verteporfin (VP) inhibits YAP-TEAD and human retinoblastoma cell growth in vitro without light activation.

Verteporfin (VP), a benzoporphyrin derivative, is clinically used in photodynamic therapy for neovascular macular degeneration. Recent studies indicate that VP may inhibit growth of hepatoma cells without photoactivation through inhibition of YAP-TEAD complex. In this study, we examined the effects of VP without light activation on human retinoblastoma cell lines. Verteporfin but not vehicle control inhibited the growth, proliferation and viability of human retinoblastoma cell lines (Y79 and WERI) in a dose-dependent manner and was associated with downregulation of YAP-TEAD associated downstream proto-oncogenes such as c-myc, Axl, and surviving. In addition VP affected signals involved in cell migration and angiogenesis such as CTGF, cyr61, and VEGF-A but was not associated with significant effect on the mTOR/autophagy pathway. Of interest the pluripotency marker Oct4 were downregulated by Verteporfin treatment. Our results indicate that the clinically used photosensitizer VP is a potent inhibitor of cell growth in retinoblastoma cells, disrupting YAP-TEAD signaling and pluripotential marker OCT4. This study highlights for the first time the role of the YAP-TEAD pathway in Retinoblastoma and suggests that VP may be a useful adjuvant therapeutic tool in treating Rb patients.

Secretory leukocyte proteinase inhibitor-competent DNA deposits are potent stimulators of plasmacytoid dendritic cells: implication for psoriasis.

Secretory leukocyte proteinase inhibitor (SLPI) is a well-established inhibitor of serine proteases such as human neutrophil elastase (HNE) and a NF-κB regulatory agent in immune cells. In this paper, we report that SLPI plays a previously uncharacterized role in regulating activation of plasmacytoid dendritic cells (pDCs). As the main source of IFN type I (IFNI), pDCs are crucial contributors to inflammatory and likely wound-healing responses associated with psoriasis. The mechanisms responsible for activation of pDCs in psoriatic skin are therefore of substantial interest. We demonstrate that in lesional skin of psoriasis patients, SLPI together with its enzymatic target HNE and DNA, is a component of neutrophil extracellular traps (NETs). Whereas SLPI(+) neutrophils and NETs were found to colocalize with pDCs in psoriatic skin, a mixture of SLPI with neutrophil DNA and HNE induced a marked production of IFNI by pDCs. IFNI synthesis by stimulated pDCs was dependent on intracellular DNA receptor TLR9. Thus, SLPI may contribute to psoriasis by enabling pDCs to sense extracellular DNA and produce IFNI.

Regulation of chemerin chemoattractant and antibacterial activity by human cysteine cathepsins.

Chemerin, a ligand for the G-protein coupled receptor chemokine-like receptor 1, requires C-terminal proteolytic processing to unleash its chemoattractant activity. Proteolytically processed chemerin selectively attracts specific subsets of immunoregulatory APCs, including chemokine-like receptor 1-positive immature plasmacytoid dendritic cells (pDC). Chemerin is predicted to belong to the structural cathelicidin/cystatin family of proteins composed of antibacterial polypeptide cathelicidins and inhibitors of cysteine proteinases (cystatins). We therefore hypothesized that chemerin may interact directly with cysteine proteases, and that it might also function as an antibacterial agent. In this article, we show that chemerin does not inhibit human cysteine proteases, but rather is a new substrate for cathepsin (cat) K and L. cat K- and L-cleaved chemerin triggered robust migration of human blood-derived pDC ex vivo. Furthermore, cat K- and L-truncated chemerin also displayed antibacterial activity against Enterobacteriaceae. Cathepsins may therefore contribute to host defense by activating chemerin to directly inhibit bacterial growth and to recruit pDC to sites of infection.

A disintegrin and metalloproteinase 10 regulates antibody production and maintenance of lymphoid architecture.

A disintegrin and metalloproteinase 10 (ADAM10) is a zinc-dependent proteinase related to matrix metalloproteinases. ADAM10 has emerged as a key regulator of cellular processes by cleaving and shedding extracellular domains of multiple transmembrane receptors and ligands. We have developed B cell-specific ADAM10-deficient mice (ADAM10(B-/-)). In this study, we show that ADAM10 levels are significantly enhanced on germinal center B cells. Moreover, ADAM10(B-/-) mice had severely diminished primary and secondary responses after T-dependent immunization. ADAM10(B-/-) displayed impaired germinal center formation, had fewer follicular Th cells, decreased follicular dendritic cell networks, and altered chemokine expression in draining lymph nodes (LNs). Interestingly, when spleen and LN structures from immunized mice were analyzed for B and T cell localization, tissues structure was aberrant in ADAM10(B-/-) mice. Importantly, when ADAM10-deficient B cells were stimulated in vitro, they produced comparable Ab as wild type B cells. This result demonstrates that the defects in humoral responses in vivo result from inadequate B cell activation, likely because of the decrease in follicular Th cells and the changes in structure. Thus, ADAM10 is essential for the maintenance of lymphoid structure after Ag challenge.

Secretory leukocyte protease inhibitor modulates FcεRI-dependent but not Mrgprb2-dependent mastocyte function in psoriasis.

Psoriasis, which involves mast cells, is a chronic inflammatory skin disorder whose pathophysiology is still not fully understood. We investigated the role of secretory leukocyte protease inhibitor (SLPI), a potential inhibitor of mastocyte serine proteases, on mast cell-dependent processes of relevance to the skin barrier defense in psoriasis. Here, we demonstrate that the dermal mast cells of patients with psoriasis express SLPI but not those of healthy donors. Moreover, SLPI transcripts were found to be markedly upregulated in murine mast cells by mediators derived from psoriasis skin explant cultures. Using mast cells from SLPI-deficient mice and their SLPI+ wild-type controls, we show that SLPI inhibits the activity of serine protease chymase in mastocytes. SLPI was also found to enhance the degranulation of mast cells activated via anti-IgE Abs but not Mrgprb2 ligands. Finally, we demonstrate that the expression and function of Mrgprb2 in mast cells are suppressed by a normal and, to a larger extent, psoriatic skin environment. Together, these findings reveal mechanisms underlying FcεRI- and Mrgprb2-dependent mast cell function that have not been described previously.

Mitochondrial-targeted Signal transducer and activator of transcription 3 (STAT3) protects against ischemia-induced changes in the electron transport chain and the generation of reactive oxygen species.

Expression of the STAT3 transcription factor in the heart is cardioprotective and decreases the levels of reactive oxygen species. Recent studies indicate that a pool of STAT3 resides in the mitochondria where it is necessary for the maximal activity of complexes I and II of the electron transport chain. However, it has not been explored whether mitochondrial STAT3 modulates cardiac function under conditions of stress. Transgenic mice with cardiomyocyte-specific overexpression of mitochondria-targeted STAT3 with a mutation in the DNA-binding domain (MLS-STAT3E) were generated. We evaluated the role of mitochondrial STAT3 in the preservation of mitochondrial function during ischemia. Under conditions of ischemia heart mitochondria expressing MLS-STAT3E exhibited modest decreases in basal activities of complexes I and II of the electron transport chain. In contrast to WT hearts, complex I-dependent respiratory rates were protected against ischemic damage in MLS-STAT3E hearts. MLS-STAT3E prevented the release of cytochrome c into the cytosol during ischemia. In contrast to WT mitochondria, ischemia did not augment reactive oxygen species production in MLS-STAT3E mitochondria likely due to an MLS-STAT3E-mediated partial blockade of electron transport through complex I. Given the caveat of STAT3 overexpression, these results suggest a novel protective mechanism mediated by mitochondrial STAT3 that is independent of its canonical activity as a nuclear transcription factor.

Soluble mediators in the function of the epidermal-immune-neuro unit in the skin.

Skin is the largest, environmentally exposed (barrier) organ, capable of integrating various signals into effective defensive responses. The functional significance of interactions among the epidermis and the immune and nervous systems in regulating and maintaining skin barrier function is only now becoming recognized in relation to skin pathophysiology. This review focuses on newly described pathways that involve soluble mediator-mediated crosstalk between these compartments. Dysregulation of these connections can lead to chronic inflammatory diseases and/or pathologic conditions associated with chronic pain or itch.

Chemerin-Derived Peptide Val66-Pro85 Is Effective in Limiting Methicillin-Resistant S. aureus Skin Infection.

Chemerin-derived peptide Val66-Pro85 (p4) restricts the growth of a variety of skin-associated bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). To better understand the antimicrobial potential of chemerin peptide, we compared p4 activity against MRSA in vitro to cathelicidin LL-37, one of the key endogenous peptides implicated in controlling the growth of S. aureus. The efficacy of p4 was also validated in relevant experimental models of skin pathology, such as topical skin infection with community-acquired MRSA, and in the context of skin inflammatory diseases commonly associated with colonization with S. aureus, such as atopic dermatitis (AD). We showed that p4 collaborates additively with LL-37 in inhibiting the growth of S. aureus, including MRSA, and that p4 was effective in vivo in reducing MRSA burden. p4 was also effective in reducing levels of skin-infiltrating leukocytes in S. aureus-infected AD-like skin. Taken together, our data suggest that p4 is effective in limiting S. aureus and, in particular, MRSA skin infection.

Redox Active Antimicrobial Peptides in Controlling Growth of Microorganisms at Body Barriers.

Epithelia in the skin, gut and other environmentally exposed organs display a variety of mechanisms to control microbial communities and limit potential pathogenic microbial invasion. Naturally occurring antimicrobial proteins/peptides and their synthetic derivatives (here collectively referred to as AMPs) reinforce the antimicrobial barrier function of epithelial cells. Understanding how these AMPs are functionally regulated may be important for new therapeutic approaches to combat microbial infections. Some AMPs are subject to redox-dependent regulation. This review aims to: (i) explore cysteine-based redox active AMPs in skin and intestine; (ii) discuss casual links between various redox environments of these barrier tissues and the ability of AMPs to control cutaneous and intestinal microbes; (iii) highlight how bacteria, through intrinsic mechanisms, can influence the bactericidal potential of redox-sensitive AMPs.